Method for activating AMPK and the use of adenine

ABSTRACT

The present invention relates to adenine which is useful to activate AMP-activated protein kinase (AMPK) and the use of adenine in the prevention or treatment of conditions or disease and thereby prevent or treat conditions or diseases which can be ameliorated by AMPK in a mammal.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to adenine which is useful to activateAMP-activated protein kinase (AMPK) and the use of adenine in theprevention or treatment of conditions or disease.

2. Description of Related Art

Adenosine 5′-monophosphate-activated protein kinase (AMPK) is a cellularenergy sensor and a responder to energy demand. AMPK is a heterotrimercomposed of catalytic α subunit and regulatory β, γ subunits. All thesesubunits are highly conserved in eukaryotes. The activation of AMPK isthrough phosphorylation on the conserved 172^(th)-threonine residue of αsubunit by upstream kinases such as LKB1, Ca²⁺/Calmodulin dependentkinase, and TAK1. High AMP/ATP ratio caused by physiological orpathological stress activates AMPK. Upon activation, AMPK activatescatabolic pathway and inhibits anabolism which in term restores cellularenergy balance by decreasing ATP consumption and promoting ATPgeneration.

As a regulator of energy homeostasis, AMPK has been suggested to be apotential drug target for metabolic syndromes including type IIdiabetes, cardio-vascular disease, and fatty liver disease. Many of themetabolic syndromes are linked to insulin resistance. Insulin resistanceis a pathological condition in which cells fail to respond to insulinthus excess glucose in the blood stream cannot be removed into skeletalmuscle or fat tissue. The activation of AMPK increases protein level ofGLUT4, a glucose transporter, via transcriptional regulation and inducesGLUT4 translocation to the plasma membrane in muscle cells in an insulinindependent manner resulting in increases in the rate of cellularglucose uptake. Activation of AMPK also inhibits fatty acids andcholesterol synthesis via suppressing acetyl-CoA carboxylase and HMG-CoAreductase, respectively. In addition, activation of AMPK leads toinhibition of several transcription factors, including SREBP-1c, ChREBPand HNF-4a, and down-regulates the expression of enzymes which aremainly involved in fatty acid synthesis and gluconeogenesis. Thesefindings support the idea that AMPK is a target of choice in thetreatment of metabolic syndrome, in particular, diabetes.

In addition to the regulation of energy homeostasis, AMPK has beenimplicated in modulating several cellular mechanisms includinginflammation, cell growth, apoptosis, autophagy, senescense anddifferentiation. Extensive studies demonstrate AMPK is a repressor ofinflammation. Activation of AMPK can inhibit inflammation viasuppressing NF-κB signaling. NF-κB signaling is the principle pathwaythat activates innate and adaptive immunity. The activation of AMPK caninhibits NF-κB transcriptional activity indirectly via stimulatingSIRT1, Forkhead box O (FoxO) family or peroxisome proliferator-activatedreceptor co-activator 1α (PGC1α). Several groups also demonstrate thatactivation of AMPK suppresses protein expression of cyclooxygenase-2(COX-2). COX-2 is an inducible enzyme which controlled bypro-inflammatory cytokines and growth factors. COX-2 convertsarachidonic acid into prostaglandin which results in inflammation andpain. Inhibition of COX-2 activity or expression has been linked toanti-inflammation.

Several AMPK activators have been demonstrated to possessanti-inflammatory function in vivo. For example,5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) has been shown toameliorate acute and relapsing colitis mouse model induced by2,4,6-trinitrobenzene sulfonic acid (TNBS) or dextran sulfate sodium.AICAR treated mice showed reduced body weight loss and significantattenuation of inflammation. AICAR also showed therapeutic effects intreating experimental autoimmune encephalomyelitis (EAE), an animalmodel of multiple sclerosis and decreases severity of LPS-induced lunginjury in mice.

Dysregulation of cellular signaling pathway can lead to abnormal cellgrowth and ultimately, cancer. The mammalian target of rapamycin (mTOR)is a serine/threonine kinase which regulates cell proliferation andautophagy. The activity of mTOR signaling pathway is dys-regulated inmany different cancers and therefore mTOR inhibitors are considered aspotential drugs for cancer therapy. There are extensive studiesdemonstrate that AMPK phosphorylates tuberous sclerosis complex 2 (TSC2)and Raptor to inhibit mTOR pathway. A variety of AMPK activatorsincluding AICAR, metformin, phenformin has also been demonstratedsuppressed mTOR signaling and inhibited cancer cell growth. In addition,activation of AMPK induces autophagy via suppressing mTORC1 activity.Due to the inhibition of mTORC1 by AMPK, phosphorylation of Ulk1 onSer⁷⁵⁷ is decreased and subsequently Ulk1 can be phosphorylated by AMPKon Ser³¹⁷ and Ser⁷⁷⁷. The AMPK-phosphorylated Ulk1 is active and theninitiates autophagy.

Base on above mentioned, AMPK has been suggested as a good target inmany human diseases or pathological conditions including inflammatorydisease, wound healing, neurodegeneration, cancer, oxidative stress andcardiovascular disease. In fact, AMPK activators have been applied forclinical trials in at least 24 disease categories including bacterialand fungal diseases, behaviors and mental disorders, blood and lymphconditions, cancers and other Neoplasms, digestive system diseases,diseases and abnormalities at or before birth, ear, Nose, and throatdiseases, eye diseases, gland and hormone related diseases, heart andblood diseases, immune system diseases, mouth and tooth diseases,muscle, bone, and cartilage diseases, nervous system diseases,nutritional and metabolic diseases, occupational diseases, parasiticdiseases, respiratory tract (lung and bronchial) diseases, skin andconnective tissue diseases, substance related disorders, symptoms andgeneral pathology, urinary tract, sexual organs, and pregnancyconditions, viral diseases, wounds and injuries. Herein we disclosed anovel AMPK activator, adenine and the use of this compound in theprevention or treatment of diseases.

BRIEF SUMMARY OF THE INVENTION

According to one embodiment of the present invention there is provided anovel AMPK activator, adenine, for activating AMPK in cells and therebyprevent or treat conditions or diseases which can be ameliorated by AMPKin a mammal.

According to one embodiment of the present invention there is provided amethod for reducing blood glucose via activating AMPK in a cell andthereby prevent or treat diseases including metabolic syndrome, type 2diabetes, insulin resistance wherein an effective amount of adenineand/or the pharmaceutically acceptable salts thereof, is administratedto a mammal in need of such treatment.

According to one embodiment of the present invention there is provided amethod for anti-inflammation via activating AMPK in a cell and therebytreat inflammatory condition or disease, wherein an effective amount ofadenine and/or the pharmaceutically acceptable salts thereof, isadministrated to a mammal in need of such treatment.

According to one embodiment of the present invention there is provided amethod for reducing Aβ accumulation via activating AMPK in a cell andthereby prevent or treat Alzheimer's disease, wherein an effectiveamount of adenine and/or the pharmaceutically acceptable salts thereof,is administrated to a mammal in need of such treatment.

According to one embodiment of the present invention there is provided amethod for suppressing fibroblast proliferation via activating AMPK andthereby prevent scar formation during wound healing.

According to one embodiment of the present invention there is provided amethod to enhance wound healing, wherein an effective amount of adenineand/or the pharmaceutically acceptable salts thereof, is administratedto a mammal in need of such treatment.

According to one embodiment of the present invention there is provided amethod to inhibit ROS production in a cell and thereby protect or treatcells from ROS injury in mammal, wherein an effective amount of adenineand/or the pharmaceutically acceptable salts thereof, is administratedto a mammal in need of such treatment.

According to one embodiment of the present invention there is provided amethod to inhibit cancer cells proliferation and thereby prevent ortreat cancer, wherein an effective amount of adenine and/or thepharmaceutically acceptable salts thereof, is administrated to a mammalin need of such treatment.

Thus, the present invention relates to adenine which is useful toactivate AMP-activated protein kinase (AMPK) and the use of adenine inthe prevention or treatment of diseases, including pre-diabetes, insulinresistance, type 2 diabetes, metabolic syndrome, obesity, inflammation,wound healing, Alzheimer's disease, cancer, oxidative stress andcardiovascular disease.

BRIEF DESCRIPTION OF THE DRAWINGS

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DETAILED DESCRIPTION OF THE INVENTION

The inventors have unexpectedly discovered that adenine is a novel AMPKactivator and has various biological functions in mammals. In recentyears, the activation of AMPK has been shown to be beneficial in theprevention and the treatment of diseases such as pre-diabetes, insulinresistance, type 2 diabetes, metabolic syndrome, obesity, inflammation,Alzheimer's disease, cancer, oxidative stress and cardiovascular diseaseas well as enhancing wound healing. The inventors contemplate that sucheffects can be attributed to the activation of AMPK which result in butnot limited to the reduction of COX-2, ROS production and increase ofglucose uptake.

As used herein, the term “AMPK” refers to adenosine5′-monophosphate-activated protein kinase. The term “AMPK activator”used herein refers to a compound which can enhance or stabilizephosphorylation of 172^(th)-threonine residue of subunit of AMPK andhence activates AMPK activity.

Contemplated Indications

Base on the inventor's findings (see examples below), it's contemplatedthat adenine can be used as a therapeutic agent for various conditionsor diseases via activating AMPK. The following provides exemplaryguidance and evidence on contemplated indications.

Adenine in the Treatment of Hyperglycemia, Pre-Diabetes, InsulinResistance, and Type 2 Diabetes

It has recently been reported that AMPK activators including metformin,A769662, AICAR reduced plasma glucose in diabetic or obesity micemodels. In the present invention, 1 μM˜600 μM of adenine significantlyincreased glucose uptake of C2C12 muscle cells (Table 2). To furtherevaluate the effects of adenine on the modulation of plasma glucoselevel, the high-fat diet-fed mice were served as a type 2 diabetesanimal model. Chronic treatment of high-fat diet-fed mice with adeninesignificantly reduced plasma glucose by more than 30% and decreasedplasma triacylglycerides by more than 35% compared to the control mice.A more-than-15% decrease in body weight was also observed (example 3).

As used herein, “hyperglycemia” refers to physiological conditioncharacterized by blood sugar higher than 126 mg/dL. “pre-diabetes”refers to a physiological condition characterized by a fasting bloodsugar higher than 100 mg/dL but below than 140 mg/dL. “Insulinresistance” used herein refers to a physiological condition in whichwhole body or tissues including liver, skeletal muscle, adipose tissuefail to response to insulin. “type 2 diabetes” used herein also known asnoninsulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes.It refers to a metabolic disorder caused by insufficient insulinproduction or insulin resistance which often manifested by a fastingglucose higher than 140 mg/dL. According to the examples, adenine wasfound to accelerate glucose uptake, therefore was suggested as a usefultreatment to the conditions or diseases which associated with high bloodglucose.

Adenine in the Treatment of Inflammation

Various AMPK activators have been demonstrated possess anti-inflammatoryfunction in vivo. For example, daily treatment of5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) in2,4,6-trinitrobenzene sulfonic acid (TNBS) or dextran sulfatesodium-treated mice ameliorates acute and relapsing colitis by reducedbody weight loss and significant attenuation inflammation. Treatment ofAICAR had therapeutic effects in experimental autoimmuneencephalomyelitis (EAE), an animal model of multiple sclerosis.Treatment of AICAR to mice decreased severity of LPS-induced lunginjury. In the present invention, adenine inhibited LPS-inducedinflammation in vitro: under LPS stimulation, the secretion level ofpro-inflammatory cytokines including TNFα, IL-1β and IL-6 weresignificantly reduced in adenine treated macrophages compared to controlcells. Adenine also decreased COX-2 expression which was induced by LPSin human macrophages (example 4). In TNBS-induced inflammatory boweldisease (IBD) mice model chronic treatment with adenine significantlyreduced pro-inflammatory cytokines including TNF, INFγ and IL-17 incolon compare to control mice and rescued body weight loss in these mice(example 5).

As used herein, “pro-inflammatory cytokines” refers to cytokines whichpromote systemic inflammation. “Inflammatory diseases” used hereinrefers to diseases associate with inflammation including but not limitedto ankylosing spondylitis, arthritis (osteoarthritis, rheumatoidarthritis (RA), psoriatic arthritis), asthma, atherosclerosis, Crohn'sdisease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis,irritable bowel syndrome (IBS), systemic lupus erythematous (SLE),nephritis, Alzheimer's, Parkinson's disease and ulcerative colitis.“COX-2” used herein refers to cyclooxygenase 2 which convertsarachidonic acid into prostaglandin. Recently, several reportsdemonstrate that AMPK is an upstream regulator of COX-2 and suppressesCOX-2 protein expression. The same with previous findings, we found thata novel AMPK activator, adenine can also suppress COX-2 expression whichsuggest adenine may be a useful compound to inhibit COX-2 mediatedinflammation. According to the present invention, adenine was found tobe able to inhibit inflammation, therefore was suggested as a usefultreatment to the conditions or diseases which associated withinflammation.

Adenine in Wound Healing and Scar Formation

AMPK has been suggested to promote cell motility and enhance woundhealing in cultured cells. An AMPK activator, resveratrol, has beenfound to enhance incisional wound healing. In addition to closing thewound, reducing scar formation during the healing process has been apreferred goal in modern medicine. Neonatal wound healing, unlike adultwound healing, does not accompany scar formation. The difference is inCox-2 activation. In adult wound healing, COX-2 activity will beelevated (by TGF-beta), resulting in the increased production ofprostaglandin at wound sites. Prostaglandin promotes fibroblast growthand collagen formation, two factors that lead to scar formation. Hence,inhibition of COX-2 activities has been considered as effectivetreatment in preventing scar formation. In the present invention,adenine inhibited human fibroblast cell growth (example 8) and reduceCOX-2 expression. Using animal model, topical treatment of adenine atwound site enhanced not only wound closure but also reduced scarformation (example 9). According to above data, topical administrationof adenine is useful to enhance would healing and prevent scarformation.

Neurodegeneration

Defects in several different cellular mechanisms has been linked toneurodegeneration, including inflammation, intracellular trafficking,and autophagy. Autophagy functions to remove dysfunctional organelles orprotein aggregates in the cell and play a crucial part in maintainingcellular homeostasis. Pathogenesis of many neurodegenerative diseasesinvolves the presence of intracellular or extracellular proteinaggregate deposits. Removal of these protein aggregates has been shownto ameliorate the progression of these diseases. Impaired autophagypathway or removal of proteins responsible for autophagy has been linkedto neurodegeneration.

AMPK activation has been shown to facilitate autophagy pathway.Therefore, promotion of autophagy pathways via activation of AMPK may bea useful strategy to prevent or control neurodegeneration. AMPKactivators have been shown to decrease amyloid deposition via autophagypathway. Daily resveratrol administration increases life span in AD micemodels. Another AMPK activator, curcumin, has also been demonstrated asa potential drug for AD therapy. In the present invention, we found thatadenine significantly enhanced autophagy activity and reduced Aβaccumulation in a dose-dependent manner in Neuro2A cells and improvedcognitive function in AD mice model (example 6 and 7). According tothese findings, adenine can be useful in the treatment ofneurodegenerative diseases.

As used herein, “neurodegeneration” refers to the condition which isprogressive loss of structure or function of neurons. Neurodegenerativedisease is a result of neurodegenerative processes including Alzheimer'sdisease (AD), Parkinson's disease (PD), Huntington's disease (HD),amyotrophic lateral sclerosis (ALS), Spinocerebellar ataxia (SCA),Spinal muscular atrophy (SMA), etc.

ROS Associated Diseases

Reactive oxygen species (ROS) including superoxide radicals, hydroxylredical and hydrogen peroxide are continuously produced in tissues. Avariety of diseases have been associated with excessive ROS includingNARP (neurogenic muscle weakness, ataxia and retinitis pigmentosa),MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-likeepisodes), MERRF (myoclonic epilepsy and ragged-red fibers), LHON (Leberhereditary optic neuropathy), and KSS (Kearns-Sayre syndrome,ophthalmoplegia, ataxia, retinitis pigmentosa, cardiac conduction defectand elevated cerebrospinal fluid protein), Parkinson disease (PD),Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS),Huntington's disease (HD), and Friedreich's ataxia (FA) and aging.Numerous reports demonstrate that AMPK activator, AICAR, reduced ROSproduction under high-glucose, palmitate or albumin induction. In thepresent invention, adenine was found to reduce ROS production in adose-dependent manner in HUVEC cell (Table 4), therefore was suggestedas a useful treatment to the conditions or diseases which associatedwith ROS.

Cancer

Activation of AMPK suppressed COX-2 and mTOR pathways which areimportant mechanisms of cancer cells growth. Due to the contribution ofmTOR and COX-2 to cancer aggressiveness, activation of AMPK is suggestedas a rational strategy for cancer therapy. Indeed numerous reports havedemonstrated that AMPK activators interrupt cancer progression. Forexample, biguanide AMPK activators (phenformin and metformin) have beenfound to inhibit breast tumor development and growth in xenografts micemodels. In the present invention, adenine was found to inhibitproliferation of human hepatocellular carcinoma cell line Hep G2, humanbreast adenocarcinoma cell line MCF7 and human colon adenocarcinomagrade II cell line HT29 (example 11). The IC50 of adenine for HepG2,MCF7 and HT29 were 544.1, 537.5 and 531.9 μM, respectively. In HepG2transplanted mice, chronic treatment with adenine significantly delayedtumor growth in dose-dependent manner. According to present invention,the treatment with adenine to activate AMPK activity may prevent orcontrol cancer development and progression.

EXAMPLE 1 AMPK Activation Assay

Effects of adenine on AMPK activation were evaluated based on thephosphorylation of AMPK protein upon adenine treatment. Mouse musclecell C2C12, mouse fibroblast 3T3, human liver carcinoma cell Hep G2,human umbilical vein endothelial cell HUVEC, Human acute monocyticleukemiacell THP1, human macrophage cell U937, murine microglia cellBV-2, and mouse neuroblastoma cell Neuro2A were cultured in high-glucoseDulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovineserum (FBS), 4 mM L-glutamine, 2 mM sodium pyruvate and 1%penicillin/streptomycin (Invitrogen GibcoBRL, Carlsbad, Calif., USA) at37° C. under 5% CO₂. Cells were plated at 3×10⁵ per well in 6-wellplates. 24 h after plating, adenine was added to the culture media asindicated. After 30 min cells were lysed and subject to western blotanalysis. Equal amount of protein from each sample was separated bySDS-PAGE and then electroblotted on to PVDF membranes. Membranes wereblocked with 3% BSA in PBS for 60 min and incubated with ananti-phospho-AMPK (Thr172) antibody (1:2,000, Cell signaling) or ananti-AMPK antibody (1:2,000, Cell signaling) at 4° C. overnight followedby the corresponding secondary antibody for 1 h at room temperature(RT). Immunoreactive bands were detected by enhanced chemiluminescence(ECL; Pierce, Rockford, Ill., USA) and recorded using Kodak film(Rochester, N.Y., USA). The detected signals were scanned and thenquantified using TotalLab Quant software (TotalLab).

The effect of adenine on AMPK activation is summarized in Table 1.Adenine significantly activated AMPK in all tested cells includingC2C12, 3T3, Hep G2, MCF7, HT29, THP1, HUVEC, U937, BV2, Dermal Papillaand Neuro2A cells.

TABLE 1 Concentration of AMPK activation Cells adenine (microM) (fold tocontrol) C2C12 1 1.2 10 1.7 100 3.2 200 3.9 600 4.1 3T3 1 1.1 10 1.5 1002.9 200 4.0 600 4.2 Hep G2 1 1.1 10 2.1 100 3.3 200 3.8 600 4.2 MCF7 11.2 10 1.6 100 2.5 200 3.4 600 3.7 HT29 1 1.1 10 1.7 100 2.9 200 3.4 6003.8 HUVEC 1 1.2 10 1.9 100 3.2 200 3.9 600 4.1 THP1 1 1.2 10 2.2 100 3.7200 4.3 600 4.2 U937 1 1.1 10 1.3 100 2.9 200 3.7 600 4.0 BV-2 1 1.2 101.7 40 2.6 160 3.2 Neuro2A 1 1.2 10 2.1 100 3.4 Dermal 1 1.1 Papilla 101.4 100 2.1 200 2.5 600 2.8

EXAMPLE 2 Glucose Uptake In Vitro

Effects of adenine on glucose uptake were analyzed by measuring theuptake of fluorescent glucose analog (2-NBDG, Molecular Probes) inmuscle cell C2C12. C2C12 were treated with indicated concentrations ofadenine for 30 min at 37° C. then incubated with 500 μM of fluorescentglucose analog. After 5 min incubation at room temperature, cells werewashed three times with Kreb-Hepes buffered solution and fixed in 70%alcohol. The fluorescence of glucose analog in cells was measured usinga Fluorescence Microplate Reader System at 480-nm excitation and 530-nmemission wavelength.

The effect of adenine on glucose uptake is summarized in Table 2.Adenine significantly stimulated glucose uptake in C2C12 cells indose-dependent manner. Data are presented as the mean±SEM of threeindependent experiments.

TABLE 2 Concentration Glucose uptake Agent (microM) (% to control)Adenine 1 117 ± 8.1  10 261 ± 13.4 100 315 ± 11.9 600 338 ± 16.5

EXAMPLE 3 Anti-Diabetic Effects of Adenine

To further evaluate the effects of adenine on the modulation of plasmaglucose level, the high-fat diet-fed mice were served as a type 2diabetes animal model. C57BL/6J mice were maintained at 22° C. under a12-h light/dark cycle and fed either a high fat diet (60% kcal % fat) ora normal diet ad libitum. Intraperitoneal injections of adenine (0.1 to50 mg/kg) or vehicle were given to the high-fat diet-fed mice from theage of 24 weeks and glucose readings were measure at 1 and 3 hr. IPadministration of adenine or vehicle only to the high-fat diet-fed micecontinued twice a day for 6 days. On day 6, plasma was collected 1 hrafter the last dosing for analyzing, evaluating, measuring plasmaglucose and triglycerides. Adenine-injected group showed >30% lowerplasma glucose and more than 35% lower plasma triacylglycerides as wellas more than 15% lower body weight compare to the control group.

EXAMPLE 4 Adenine Suppressed Inflammatory Response Induced by LPS InVitro

Effects of adenine on inflammatory response were evaluated in human THP1macrophage by examining protein level of intracellular COX-2 andsecreted TNFα, IL-1β and IL-6. Differentiation of THP1 monocytes intomacrophages was induced by 50 nM PMA for 24 hr. THP1 macrophages werefurther stimulated by 50 ng LPS for 6 hr in the presence of 10˜600 μM ofadenine or vehicle followed by cell lysis and western blot analysis.Equal amounts of protein were separated by SDS-PAGE and thenelectroblotted on to PVDF membranes. Membranes were blocked with 3% BSAin PBS for 60 min and incubated with an anti-COX-2 antibody (1:1,000,Cell signaling), an anti-actin antibody (1:5,000, Cell signaling) at 4°C. overnight followed by the corresponding secondary antibody for 1 h atroom temperature (RT). Immunoreactive bands were detected by enhancedchemiluminescence (ECL; Pierce, Rockford, Ill., USA) and recorded usingKodak film (Rochester, N.Y., USA). The detected signals were scanned andthen quantified using TotalLab Quant software (TotalLab). The secretedTNFα, IL-1β and IL-6 were analyzed by enzyme-linked immunosorbentassays. The expression of COX-2 and secretion level of TNFα, IL-1β andIL-6 were significantly reduced in adenine treated macrophages comparedwith control cells.

TABLE 3 Adenine TNF.(% to IL-1β.(% to IL-6(% to COX-2 (% to (microM)control) control) control) control) 0 100 ± 4.7  100 ± 11.3 100 ± 8.5 100 ± 2.9  10 85 ± 9.1 91 ± 8.4 88 ± 6.3 81 ± 4.4 100 41 ± 2.6 29 ± 5.521 ± 7.8 59 ± 3.5 600 23 ± 1.8 17 ± 3.7 14 ± 6.2 38 ± 5.3

EXAMPLE 5 Adenine Suppressed 2,4,6-Trinitrobenzene Sulfonic Acid (TNBS)Induced Inflammation In Vivo

To further evaluate the effects of adenine on inflammatory response,TNBS-induced inflammatory bowel disease (IBD) mice model was used.C57BL/6J mice were maintained at 22° C. under a 12-h light/dark cycle.Relapsing colitis was induced with five escalating doses of TNBS, 0.5mg, 0.75 mg, 1.0 mg, 1.25 mg, and 1.5 mg, in 50% ethanol wereadministered respectively for 0.1 mL per mouse weekly. Control mice weregiven 0.1 mL of saline alone by intrarectal administration. After thethird administration of TNBS, daily intraperitoneal injection of adenine(0.01, 0.1, 5 or 30 mg/kg body weight) or vehicle were given to mice.Two days after the last TNBS administration, mice were sacrificed. Theinflammatory cytokines including TNF, INFγ and IL-17 from coloniclysates were evaluated by enzyme-linked immunosorbent assays. The levelof TNF, INFγ and IL-17 were significantly reduced in colon of adeninetreated mice in dose-dependent manner compared with non-adenine treatedmice. In addition, treatment of adenine also rescued the body weightloss caused by TNBS.

EXAMPLE 6 Amyloid β Peptide and Autophagy Assay

Effects of adenine on Amyloid β peptide were analyzed in mouseneuroblastoma cell Neuro2A. Neuro2A cells were cultured in high-glucoseDulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovineserum (FBS), 4 mM L-glutamine, 2 mM sodium pyruvate and 1%penicillin/streptomycin (Invitrogen GibcoBRL, Carlsbad, Calif., USA) at37° C. under 5% CO₂. Cells were plated at 3×10⁵ per well (6-well plate).24 h after plating, cells were transfected with human APP695 and treatedwith indicated concentration of adenine for 24 h followed by cell lysisand western blot analysis. Equal amounts of protein were separated bySDS-PAGE and then electroblotted on to PVDF membranes. Membranes wereblocked with 3% BSA in PBS for 60 min and incubated with an anti-Aβantibody (1:1,000, abcam), an anti-LC3 antibody (1:1,000, Cellsignaling), and an anti-actin antibody (1:5,000, Cell signaling) at 4°C. overnight followed by the corresponding secondary antibody for 1 h atroom temperature (RT). Immunoreactive bands were detected by enhancedchemiluminescence (ECL; Pierce, Rockford, Ill., USA) and recorded usingKodakfilm (Rochester, N.Y., USA). The detected signals were scanned andthen quantified by using TotalLab Quant software (TotalLab).

The effects of adenine on sAβ production and LC3-II/LC3-I ratio aresummarized in Table 4. Adenine significantly reduced Aβ amount andincreased LC3-II/LC3-I ratio in dose-dependent manner in Neuro2A cells.Because the conversion of LC3-I to LC3-II is indicative of autophagyactivity, the higher LC3-II/LC3-I ratio in adenine treated cellsreflects the ability of adenine to induce autophagy activity.

TABLE 4 Adenine sAβ level LC3-II/LC3-Iratio (microM) (% of control)(relative to control) 0  100 ± 6.1 1.0 ± 0.1 10  89 ± 7.5 1.2 ± 0.1 20 63 ± 2.2 1.8 ± 0.3 30 48.1 ± 1.7 2.8 ± 0.2 40 31.7 ± 5.1 2.9 ± 0.2 5029.4 ± 3.6 3.2 ± 0.3

EXAMPLE 7 Adenine Rescued Neurodegeneration of Aβ-Induced Alzheimer'sDisease Model Mice

Aβ25-35 was purchased from Sigma-Aldrich (St. Louis, Mo.). The peptideswere dissolved in distilled saline and aggregated by incubation at 37°C. for 7 days before injection. C57BL/6J mice were maintained at 22° C.under a 12-h light/dark cycle. Adult mice were anesthetized by ketamine(500 mg/kg) and xylazine (100 mg/kg) and placed in a stereotaxic frame(Stoelting, Wood Dale, Ill., USA). 5 nmol of the aggregated Aβ25-35 wereinjected into the lateral ventricle using a 10-μl Hamilton syringe(Hamilton Company, Reno, Nev., USA). The target anterior-posterior (AP),medial-lateral (ML) and dorsoventral coordinates were −0.5 mm, ±1 mm and−2.5 mm relative to the bregma. To evaluate the effect of adenine onneurodegenerative disease, Aβ infusion mice were daily intraperitoneallyinjected with 0.01, 0.1, 5 or 30 mg/kg bodyweight of adenine or vehiclefor 4 weeks. The cognitive functions of these mice were analyzed byusing Morris water maze assay after 4 weeks injection. The water mazewas performed in a circular pool filled with water and a platform wassubmerged below the water's surface in the target quadrant for hiddenplatform test. During the 5-day hidden platform test, mice were randomlyplaced into starting points of the pool in each daily trial (6 trialsper day). The probe trial was performed 1 day after 5-day hiddenplatform test. For the probe trial, the platform used in the hiddenplatform test was removed and the starting point was in the quadrantopposite the target quadrant. Mice were allowed to swim in the maze for60 s and recorded by a video camera. The latency to find the platformand swim paths were analyzed by EthoVision software (Version 3.1,Noldus, The Netherlands). In the hidden platform test, adenine treatedAD mice took significantly shorter time to find platform indose-dependent manner than control AD mice. This result demonstratedadenine treatment rescued the impairments of special learning and memoryof AD mice. Further, adenine treated AD mice spent higher percentagetime in target quadrant in probe assay than control AD mice, whichindicated that adenine improve the retention of memory.

EXAMPLE 8 Adenine Inhibited Fibroblast Proliferation

Human fibro blast cell line 3T3 were cultured in high-glucose Dulbecco'smodified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS),4 mM L-glutamine, 2 mM sodium pyruvate and 1% penicillin/streptomycin(Invitrogen GibcoBRL, Carlsbad, Calif., USA) at 37° C. under 5% CO₂. Forcell proliferation assay, 3T3 cells were plated at 1×10⁵ per well(6-well plate). 24 h after plating, cells were treated with indicatedconcentration of adenine for 72 h and the number of viable cells wascounted. Cells were detached using trypsin-EDTA solution and stainedwith trypan blue. The living cells were counted using hemocytometer. Theeffects of adenine on 3T3 cell proliferation are summarized in Table 5.Adenine significantly inhibited 3T3 cell proliferation in dose-dependentmanner. Data are presented as the mean±SEM of three independentexperiments.

TABLE 5 Adenine Cell number (microM) (% to control) 0 100 ± 4.3  10 91 ±2.7 50 73 ± 8.1 100 64 ± 5.3 200 48 ± 2.8 500 33 ± 6.4 1000  27 ± 11.3

EXAMPLE 9 Adenine Enhances Wound Healing and Reduces Scar Formation

C57BL/6J mice were maintained at 22° C. under a 12-h light/dark cycle.The experiments were performed with 12-weeks old mice. Afteranesthetized by an intraperitoneal injection of ketamine (500 mg/kg) andxylazine (100 mg/kg), 6-mm full-thickness excisional skin wounds wasmade on the backs of mice using 6-mm skin biopsy punches. Immediatelyafter wounding, 10˜1200 M of adenine in 25 l saline or saline alone wasapplied to the wound bed. The skin wounds were then covered bysemipermeable transparent dressing and fixed to the skin. The mice weretreated with adenine or vehicle for 14 days and then sacrificed. Thescar formation is assessed by Masson's trichrome staining to observe thefibrosis process and the collagen framework of the healed wound (Fixedby 4% paraformaldehyde). After 14 days of treatment, the extent ofclosure was significantly greater in adenine treated mice indose-dependent manner than in control mice. According to histologicalexamination of the regenerated tissue, topical treatment with adeninesignificantly decreased the scar width 14 days post-wounding compared tovehicle treated wounds.

EXAMPLE 10 Adenine Reduced ROS Production

HUVECs were cultured in Dulbecco's modified Eagle's medium (DMEM)containing 10% fetal bovine serum (FBS), 4 mM L-glutamine, 2 mM sodiumpyruvate and 1% penicillin/streptomycin (Invitrogen GibcoBRL, Carlsbad,Calif., USA) at 37° C. under 5% CO₂. Cells were plated at 2×10⁴ per wellin black 96-well. 24 h after plating, medium was changed to fresh DMEMcontaining either 5.6 or 30 mM glucose and treated with indicatedconcentration of adenine or vehicle. 24 hr after treatment, theintracellular ROS was detected using H₂DCF-DA. Cells were washed oncewith PBS and then incubated with 100 μM DCF at 37° C. for 30 min thenthe fluorescence of DCF was measured using a Fluorescence MicroplateReader System at 485-nm excitation and 530-nm emission wavelengths.

The effect of adenine on ROS production is summarized in Table 4.Adenine significantly reduced hyperglycemia-induced ROS production indose-dependent manner in HUVECs.

TABLE 4 Adenine Glucose ROS production (microM) (mM) (% of 5.6 mMglucose) 0 30  275 ± 8.1 10 30  211 ± 4.3 100 30  116 ± 1.7 200 30 38.1± 2.9 600 30 21.7 ± 3.1 1200 30 22.4 ± 2.5

EXAMPLE 11 Cancer Cell Growth Inhibition Assay

Human liver hepatocellular carcinoma cell line Hep G2, human breastadenocarcinoma cell line MCF7 and human colon adenocarcinoma grade IIcell line HT29 were used to evaluate the effects of adenine on cellproliferation. Those cell lines were obtained from ATCC and werecultured in high-glucose Dulbecco's modified Eagle's medium (DMEM)containing 10% fetal bovine serum (FBS), 4 mM L-glutamine, 2 mM sodiumpyruvate and 1% penicillin/streptomycin (Invitrogen GibcoBRL, Carlsbad,Calif., USA) at 37° C. under 5% CO₂. Cells were plated at 1×10⁵ per well(6-well plate). 24 h after plating, cells were treated with indicatedconcentration of adenine for 48 h and then followed by cell counting.Cells were detached using trypsin-EDTA solution and stained with trypanblue. The living cells were counted using hemocytometer. The IC50 ofadenine for HepG2, MCF7 and HT29 were 544.1, 537.5 and 531.9 μM,respectively.

EXAMPLE 12 Tumor Growth Assay

Human liver hepatocellular carcinoma cell line Hep G2 were cultured inhigh-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10%fetal bovine serum (FBS), 4 mM L-glutamine, 2 mM sodium pyruvate and 1%penicillin/streptomycin (Invitrogen GibcoBRL, Carlsbad, Calif., USA) at37° C. under 5% CO₂. For tumor implantation, 5×10⁶ Hep G2 cells wereinjected subcutaneously into 8-week-old male nonobese diabetic-severecombined immunodeficiency (NOD-SCID) mice. After implantation, the micewere daily intraperitoneally injected with 5, 20 or 50 mg/kg body weightof adenine or vehicle and the tumor size was monitored every 3 days. Thegrowth of tumor was significantly retarded in adenine treated micecompared with control mice 14 days post implantation.

It should be apparent, however, to those skilled in the art that manymore modifications besides those already described are possible withoutdeparting from inventive concepts herein. The embodiments are notintended to limit the scope of the present invention. The scope of thepresent invention is defined only by the appended claims.

The invention claimed is:
 1. A method for treating a condition selectedfrom the group consisting of pre-diabetes, type 2 diabetes, andmetabolic syndrome, comprising administrating to a mammal in needthereof with an effective amount of adenine and/or a pharmaceuticallyacceptable salt thereof to thereby increase glucose uptake into a celland thereby treat the condition, wherein the effective amount is in arange of from 0.1 mg/kg body weight to 20 mg/kg body weight.
 2. A methodfor treating Alzheimer's disease, the method comprising administratingto a mammal in need thereof with an effective amount of adenine and/or apharmaceutically acceptable salt thereof as a sole active ingredient tothereby inhibit Aβ accumulation in a cell and thereby treat theAlzheimer's disease.
 3. A method for activating AMPK to thereby increaseglucose uptake into a cell and thereby treat a condition selected fromthe group consisting of pre-diabetes, type 2 diabetes, and metabolicsyndrome, comprising administrating to a mammal in need thereof with aneffective amount of adenine and/or a pharmaceutically acceptable saltthereof, wherein the effective amount is in a range of from 1 μM to 600μM.
 4. The method of claim 2, wherein said method enhances autophagyactivity in said mammal to thereby inhibit Aβ accumulation in the cell.